This journal is© the Owner Societies 2014 Phys. Chem. Chem. Phys.

Cite this:DOI: 10.1039/c3cp55371j

Cyclic dipeptide immobilization on Au(111) andCu(110) surfaces

Oksana Plekan,*a Vitaliy Feyer,b Sylwia Ptasinska,c Nataliya Tsudd andKevin C. Princeaef

Soft X-ray Photoelectron Spectroscopy (XPS) and Near Edge X-ray Absorption Fine Structure (NEXAFS)

spectroscopy have been used to probe the electronic and adsorption properties of two cyclic

dipeptides, i.e. cyclo(glycyl-histidyl) and cyclo(phenylalanyl-prolyl), on Au(111) and Cu(110) surfaces. The

core level spectra show chemical shifts which indicate weak chemisorption on Au(111), and stronger

chemisorption on the Cu(110) surface, mainly via one of the nitrogen atoms in the central rings of both

molecules, and nitrogen in the imidazole ring of cyclo(glycyl-histidyl). From the angular dependence of

the NEXAFS spectra at the O and N K-edges, we conclude that both dipeptides have a preferred

orientation on the two surfaces.

Introduction

Immobilization of organic molecules on surfaces has beenstudied in detail over the last few decades because of itsimportance in applications in biomedical materials engineeringand bio-nano-technology.1 Understanding of the physical andchemical processes that occur during biomolecule adsorptionis needed for a priori design of surfaces to achieve a desiredresponse, for example in sensor systems or drug delivery schemes.Biomolecular adsorption is a complex process that besidescovalent bonding may involve other interactions, such as ionic,hydrogen or van der Waals bonding,2 that depend in part onmolecular structure, size, and stability.3

The molecule–surface interaction occurs via various func-tional groups that constitute a molecule, therefore the choice ofappropriate functional groups allows molecules to act as build-ing blocks in the fabrication of complicated architectures.Molecules having different terminal groups, such as sulfhydryl,silanes, carboxyl or amino groups, can be used to bind themolecule to the surface of a substrate, and be manipulated to

build up a hierarchical self-assembled structure.4–6 For example,amino acids consist of a side chain plus two important functionalgroups, i.e. –COOH and –NH2, which are available for immobi-lization on the surface. These groups may change their chargestate to a protonated amino group (–NH3

+) and a deprotonatedcarboxylate group (–COO�). If both are present in an amino acid,the zwitterions may interact ionically, while the neutral groupsmay form hydrogen bonds.

Many theoretical and experimental studies of biomoleculesare focused on the adsorption geometry and interactions ofamino acids and their polymers, i.e. polypeptides and proteins,with different metal surfaces.7–10 Recently, by using spectro-scopic methods such as valence band photoemission spectro-scopy, XPS and NEXAFS we have successfully investigated theelectronic and structural properties of monolayer and sub-monolayer films of amino acids and short peptides on Au(111),Au(110) and Cu(110) surfaces.11–14 The chemical shifts in photo-emission spectra of these molecules revealed strong interactionswith metal surfaces, indicating that chemisorption rather thanphysisorption takes place. It was shown that amino acidsinteract with surfaces mainly via the two oxygen atoms of thecarboxylate group (COO�) and functional groups containingnitrogen, i.e. imine or amine groups. Additionally, it has beenobserved that in the case of amino acids and peptides whichcontain an unsaturated ring in their structures, for exampleimidazole (IM) in histidine, the adsorption geometry of suchcompounds can vary depending on the type of metal surfaceand film thickness.11,12 The dipeptide of glycine binds to theCu(110) surface by forming two structures, zwitterionic andanionic, whose population ratio depends on the surfacecoverage.13 The anionic form of this compound binds tothe surface either via the O atoms of the carboxylate and

a Sincrotrone Trieste S.C.p.A., in Area Science Park, Strada Statale 14, km 163.5,

I-34149 Basovizza, Trieste, Italy. E-mail: oksana.plekan@elettra.trieste.it;

Fax: +39 0403758565; Tel: +39 0403758582b Peter Grunberg Institute (PGI-6) and JARA-FIT, Research Center Julich,

52425 Julich, Germanyc Radiation Laboratory and Department of Physics, University of Notre Dame,

Notre Dame, IN 46556, USAd Faculty of Mathematics and Physics, Department of Surface and Plasma Science,

Charles University, V Holesovickach 2, 18000 Prague 8, Czech Republice I.O.M.-C.N.R., in Area Science Park, Strada Statale 14, km 163.5,

I-34149 Basovizza, Trieste, Italyf Chemistry Laboratory, Faculty of Life and Social Sciences, Swinburne University of

Technology, Hawthorn, Melbourne, Victoria 3122, Australia

Received 20th December 2013,Accepted 10th February 2014

DOI: 10.1039/c3cp55371j

www.rsc.org/pccp

PCCP

PAPER

Publ

ishe

d on

10

Febr

uary

201

4. D

ownl

oade

d by

SIN

CR

OT

RO

NE

TR

IEST

E S

C. P

.A. o

n 03

/03/

2014

13:

34:2

7.

View Article OnlineView Journal

Phys. Chem. Chem. Phys. This journal is© the Owner Societies 2014

peptide group, or the amino nitrogen atom. The geometry ofthe anionic form on the surface is found to be similar to thezwitterionic one but with an additional interaction of theamino group with the surface via hydrogen atoms.13

The above studies demonstrate that well-defined systemsconsisting of simple biomolecules adsorbed on clean crystal-line surfaces can be understood in depth by applying thetechniques of modern surface science. At the other extremeare highly complex systems such as proteins adsorbed onpolycrystalline materials. A major goal of our research is toprogress from simple to increasingly complex systems, learningat each stage what level of detail we can probe in these systems.Here we have taken a step towards understanding the adsorp-tion behaviour of larger molecules, namely cyclic dipeptides.Unlike amino acids, they do not have a carboxylic acid group,but instead contain two peptide groups with a rigid geometricalrelationship. One question which arises is whether thesedipeptides bond to the surface via both peptide groups, whichwe answer below.

These biologically active materials have attracted consider-able interest15 as pharmaceuticals as they have inherentphysiological advantages, including stability (resistance toenzymatic degradation) compared to their linear counter-parts, improved receptor site selectivity and pharmacologicalspecificity.16–18 Many intrinsic properties of biomolecules aremasked by their environment or by their interactions with it.For this reason, the electronic structure of various cyclic dipeptideshave been investigated in the vapour phase where such inter-ference is absent, by means of photoemission spectroscopy andtheoretical modeling.19,20

The simplest cyclic peptide is glycine anhydride, or 2,5-diketo-piperazine (DKP), and is formed by the reaction of two glycinemolecules, with loss of water. DKP is the basis of all cyclicdipeptides, which are then differentiated by the side-chains ofthe amino acid residues forming the dipeptide.17 Although DKP isnot aromatic, this six-membered ring with two keto-groups isplanar in the solid state, except for the hydrogen atoms of themethylene groups.21 Thus some conjugation in the molecule, andpossibly the formation of bonds with a surface via the weak p-likebonds, or via the oxygen atoms, may occur.

In this work, we probe the adsorption geometry of two cyclo-peptides, cyclo(glycyl-histidyl), or c(Gly-His), and cyclo-(phenylalanyl-prolyl), or c(Phe-Pro) (see Fig. 1) immobilizedon Au(111) and Cu(110). The c(Gly-His) molecule contains anIM ring in the histidyl moiety, which can bond to the surface,

while the c(Phe-Pro) peptide contains the phenyl group and thepentagonal pyrrolidine ring fused to the DKP ring (see Fig. 1).

Experimental

The photoemission and photoabsorption spectra of cyclicdipeptides deposited on Au(111) and Cu(110) single crystalswere recorded at the Materials Science Beamline, Elettra inTrieste, using apparatus and calibration methods described indetail elsewhere.11–14,22

The c(Gly-His) and c(Phe-Pro) samples were supplied byBachem23 and used without further purification. The com-pounds were evaporated from a home-made Knudsen cell andchecked for signs of thermal decomposition, by checking forchanges in valence band spectra while heating or discolorationof the powder after heating. No evidence was found indicatingthermal decomposition of these compounds. The c(Gly-His)powder was heated to 420 K and dosed for about 10 min ontothe Au or Cu substrate. Then the sample was transferred into themain chamber and the multilayer film was flashed by heating to410 K to desorb any weakly bonded molecules. A similar proce-dure was used for c(Phe-Pro), with an evaporation temperature of360 K for 20 min, for deposition onto Au(111) and Cu(110),followed by flashing of both substrates to 370 K. The flashtemperature for both compounds was chosen to be a plateau inthe thermal desorption process, in the sense that the coverageobtained is the same for temperatures �25 K. This preparationmethodology leads to the formation of chemisorbed monolayers(saturated layers) of dipeptides on the metal substrates. Sub-sequent annealing to the same temperature did not change theratios of areas of the C 1s, N 1s and O 1s XPS signals.

The Au(111) and Cu(110) single crystal substrates, of 10 mmdiameter, were cleaned using standard procedures of cycles ofAr ion sputtering (at kinetic energy 1.0 keV) followed by flashingto 870 K. The surface order and cleanliness were monitored bylow-energy electron diffraction and XPS. Contaminants (suchas C, N, and O) were found to be below the detection limit.

The C 1s and N 1s XPS spectra were recorded in normalemission (incidence/emission angles of 601/01) using the multi-channel Specs Phoibos electron energy analyzer of the beam-line. The photon energy was 500 eV and the total resolution was0.45 eV. The O 1s core level spectra were recorded using thesame analyzer using Al Ka radiation as the ionization source,and the total energy resolution was 0.85 eV. The bendingmagnet source of the beamline had a low flux at photonenergies above the O K-edge, also partly because the beamlineoptics were contaminated with carbon, a problem which hassince been solved. The binding energy (BE) scale was calibratedby measuring the Fermi edge.

The NEXAFS spectra were taken at the N and O K-edgesusing the nitrogen and oxygen KVV Auger yield (kinetic energywindows 355–390 eV and 495–525 eV, respectively) at normal(NI, 901) and grazing (GI, 101) incidence of the photon beamwith respect to the Au(111) and Cu(110) surface. In the case ofthe Cu(110) substrate, the NEXAFS spectra were taken at

Fig. 1 Schematic structures of the dipeptides: (a) cyclo(glycyl-histidyl),and (b) cyclo(phenylalanyl-prolyl).

Paper PCCP

Publ

ishe

d on

10

Febr

uary

201

4. D

ownl

oade

d by

SIN

CR

OT

RO

NE

TR

IEST

E S

C. P

.A. o

n 03

/03/

2014

13:

34:2

7.

View Article Online

This journal is© the Owner Societies 2014 Phys. Chem. Chem. Phys.

geometries with the E-vector perpendicular or parallel to theclose-packed copper rows, i.e. along the [001] or [1%10] direction,respectively, at normal incidence, and nearly parallel to thesurface [110]-direction at grazing incidence. The polarization oflight from the beamline has not been measured, but it isbelieved to be between 80 and 90% linear, as the source is abending magnet. The energy resolution for NEXAFS measure-ments was estimated to be 0.8 eV. The raw NEXAFS data werenormalized to the intensity of the photon beam and correspondingsubstrate background adsorption spectra were subtracted.

Results and discussionCore level spectra

The thickness of layers on the crystal was estimated from theattenuation of the Au 4f7/2 and Cu 2p3/2 photoelectron signalsfrom the substrate, recorded before and after peptide deposition(Fig. 2). The Cu 2p3/2 (933 eV) level was photoionized by Al KaX-rays to give photoelectrons of energy B553 eV. In order toobtain a semi-quantitative estimate of coverage, we use theparameterized inelastic mean free path (lm) of Seah andDench24 for organic materials:

lm ¼ 49.Ek

2 þ 0:11�ffiffiffiffiffiffiEk

p;mg m�2

where Ek is the kinetic energy of the photoelectron.The resulting value was converted to length by dividing it by

the known densities of each compound,25 1.26 g cm�3 forc(Phe-Pro) and 1.41 g cm�3 for c(Gly-His). Since these densitiesare rather similar, only small variations in mean free paths areexpected. The effective film thicknesses for c(Phe-Pro) andc(Gly-His) on Au(111) were estimated to be 3.3 Å and 2.9 Å,while on Cu(110) they were 4.3 Å and 4.6 Å, respectively. Theeffective thickness is an index of how thick a layer is within acontinuum model of the overlayer; the number obtained is

essentially qualitative as there is not enough structural infor-mation at the atomic level to quantify it more precisely.

The Au 4f7/2 spectrum of the clean gold substrate has twocontributions due to atoms in the bulk (83.98 eV) and surface(83.69 eV).12,26 After adsorption of c(Gly-His) and c(Phe-Pro) onthe Au substrate, the lower energy component of the Au 4f7/2

spectrum shifted to higher BE and was not resolved from thebulk feature. The significant binding energy shift (B80 meV) ofthe surface component implies a chemical shift due to thechemisorption of the dipeptides (see Fig. 2a).26 Note that the BEshift of the surface component with respect to the clean goldsubstrate can also be due to reconstruction of the surfaces.27,28

Since alteration of the reconstruction requires a chemical inter-action, the shift is in any case indirect evidence of chemicaleffects.

The C, N and O 1s core level photoemission spectra of c(Gly-His)and c(Phe-Pro) adsorbed on Au and Cu substrates (upper panel)and in the gas phase (lower panel)19,20 are presented in Fig. 3. Thepeak positions and assignments are presented in Table 1.

The C 1s core level spectra of c(Gly-His) adsorbed on Au(111)and Cu(110) surfaces are very similar and resemble the corre-sponding spectra of linear Gly-His on Au(111).12 There are twopeaks, whereas in the gas phase, these are resolved into threepeaks. The higher BE peak A in the C 1s spectra of c(Gly-His) at288.25 eV on Au(111) and 288.30 eV on Cu(110) is assigned tocarbon atoms in the peptide N–CQO group (see Fig. 3a), a valuewhich is close to that of the BE of the carbon atom in thepeptide groups of adsorbed amino acids and peptides.11,12,29–32

The peak B at 285.85 eV is attributed to carbon atoms bondedto nitrogen (C–N) and carbon (C–C, CQC) atoms located in theimidazole side chain and DKP rings. The intensity ratio of peak Ato peak B in the C 1s spectrum of c(Gly-His) on Au and Cu is 1 : 2.8and 1 : 3.2, respectively. These numbers are in reasonable agree-ment with the expected stoichiometric ratio (A : B = 1 : 3), support-ing our assignment of the peaks in the spectra.

Fig. 2 (a) Au 4f7/2 and (b) Cu 2p3/2 core-level spectra of clean surfaces and after deposition of c(Gly-His) and c(Phe-Pro) molecules. Photon energy:120 eV and 1486.6 eV (Al Ka) for Au and Cu, respectively.

PCCP Paper

Publ

ishe

d on

10

Febr

uary

201

4. D

ownl

oade

d by

SIN

CR

OT

RO

NE

TR

IEST

E S

C. P

.A. o

n 03

/03/

2014

13:

34:2

7.

View Article Online

Phys. Chem. Chem. Phys. This journal is© the Owner Societies 2014

The carbon 1s spectra of c(Phe-Pro) adsorbed on Au(111) orCu(110) exhibit three well resolved features (see Fig. 3a) whichare consistent with the gas phase data. The high binding energypeak A is assigned to the carbonyl carbons, as in the gas phase.The maximum B in the C 1s core level spectrum (see Fig. 3a)contains contributions from different carbon atoms singlybonded to nitrogen atoms (C3, C6 and C9).19 The peak C atlow binding energy is due to photoelectrons ejected from thecarbon atoms in the Phe side-chain (see Table 1). The intensityratio A : B : C in the spectrum of c(Phe-Pro) in the gas phase is

0.14 : 0.23 : 0.63, and the solid state spectrum of c(Phe-Pro)adsorbed on Au(111) resembles the spectrum in the gasphase,19 with broadening due to solid state effects. In contrast,the corresponding spectrum on the Cu(110) surface shows astronger intensity of shoulder B (A : B : C = 0.15 : 0.33 : 0.52) withrespect to the expected stoichiometric intensity. This evidencesuggests that the local bonding of the carbon atoms of c(Phe-Pro)does not change on the Au(111) surface, in contrast with theCu(110) surface, where changes are much more pronounceddue to stronger chemisorption on this surface.

Fig. 3 C 1s (a), N 1s (b) and O 1s (c) core level spectra of c(Gly-His) and c(Phe-Pro) adsorbed on Au(111) and Cu(110). The gas phase spectra are shifted by�5.8 eV.

Table 1 C, N, and O 1s binding energies of cyclic dipeptides on the Au(111) and Cu(110) surfaces

C 1s N 1s O 1s

c(Gly-His) Au(111) 288.25 (A)285.90 (B)

400.20399.05 (shoulder)

531.23

Cu(110) 288.30 (A)285.80 (B)

400.55 (A)399.35 (B)398.40 (C)

531.65

Gas phase20 293.78 (CQO)291.88 (C–N)290.98 (C–C, CQC)

406.52 (N amino in IM)405.87 (N in DKP ring)404.57 (N imino)

536.94 (OQC)

c(Phe-Pro) Au(111) 287.48 (A)285.53 (B)284.28 (C)

399.53 531.05

Cu(110) 287.90 (A)286.10 (B)284.70 (C)

400.15 (A)398.45 (B)

531.40

Gas phase19 293.40 (CQO)291.74 (C–N)290.60 (C–C)290.27 (CQC)

405.53 (N–C) 536.95 (OQC)

Paper PCCP

Publ

ishe

d on

10

Febr

uary

201

4. D

ownl

oade

d by

SIN

CR

OT

RO

NE

TR

IEST

E S

C. P

.A. o

n 03

/03/

2014

13:

34:2

7.

View Article Online

This journal is© the Owner Societies 2014 Phys. Chem. Chem. Phys.

The N 1s spectra of the cyclic dipeptides are shown inFig. 3b. The spectra of c(Phe-Pro) and c(Gly-His) adsorbed onAu(111) resemble the spectra of these molecules in the gasphase, but with broadening due to solid state effects. The twonitrogen atoms of c(Phe-Pro) are in chemically similar environ-ments, so a small chemical shift is expected for the freemolecule and indeed, their theoretical N 1s binding energiesare separated by only 100 meV.19 The experimental N 1sspectrum of isolated c(Phe-Pro) shows a single peak, which isalso the case for c(Phe-Pro) on Au(111) but with a much broaderpeak of full width at half maximum (FWHM) B1.3 eV. Thisimplies that the two nitrogen atoms are in rather similarenvironments in the adsorbed state.

In contrast to the Au(111) surface, the N 1s core levelspectrum of c(Phe-Pro) adsorbed on Cu(110) consists of twofeatures, a strong peak at 400.15 eV (A) and a smaller peakcentred at 398.45 eV (B), with a fitted intensity ratio of 2 : 1. Weassign the higher BE peak A to species that are not chemisorbedon the copper surface, which may be stabilized by intermolecularhydrogen bonds. Peak B is assigned to chemisorption andformation of a N–Cu bond. The attribution of a low BE N 1scontribution to the interaction with copper has already beendiscussed for other peptides on copper (see for example, ref. 10,11 and 13).

Based on the observed intensity ratio we propose that underthe present experimental conditions, about 66% of c(Phe-Pro)molecules are strongly bonded via one N atom with the Cusurface, and dehydrogenation of the amino nitrogen atom mayoccur. This assignment is supported by the chemical shift tolower BE, which is observed in the N 1s spectrum of theadsorbed molecules. The species not bonded to the coppermay be stabilized on the surface by inter molecular hydrogenbonds, forming hydrogen networks, and some molecularnucleation may also occur. However we believe this is a minoreffect as the weakly bound species are mostly desorbed duringthe thermal treatment of the adlayer.

The three peaks of the N 1s spectrum of c(Gly-His) depositedon Cu were fitted with three Gaussian peaks (not shown inFig. 3b) with energy 400.55 (A), 399.35 (B) and 398.40 (C) eV.The higher BE peak A was assigned to amino nitrogen atomswhich are located in the DKP and IM rings. From a comparisonof the c(Gly-His) and c(Phe-Pro) spectra in the gas phase, weconclude that the core level of the amino nitrogen atom of theIM ring moves to higher BE compared to the amino nitrogenatoms in the DKP ring. The N 1s binding energy of the secondpeak B is about 1.2 eV lower than that of the main feature and isdue to the imino N atom in the imidazole ring.

The third peak C at 398.40 eV matches well with the energy ofpeak B in the N 1s spectrum of c(Phe-Pro) deposited on Cu(110)and we assign it to a N–Cu bond. This strong chemical shift proveschemical interaction (rather than physisorption) of c(Gly-His) onCu(110). As for the N 1s spectrum of c(Gly-His) adsorbed on gold,it contains a strong peak due to the amino nitrogen atoms and ashoulder which is associated with the imino nitrogen atom.

The O 1s core level spectra of the cyclic dipeptides adsorbedon the Au(111) and Cu(110) surfaces are shown in Fig. 3c.

The shapes of the peaks for both samples are similar to those inthe gas phase but slightly broader. A small shift of 0.4 eVtowards lower BE was observed in the case of adsorption onAu(111) with respect to Cu(110), consistent with weaker chemi-sorption. The signal in the O 1s spectra originates from the twooxygen atoms located in the DKP ring and they are locallyequivalent (see Fig. 1). From the theoretical results for the gasphase, it is known that the functional groups of the side chainsof the amino groups do not affect the DKP ring strongly, andtherefore only one broad peak was observed in the O 1s corelevel spectra.19 Maxima at 531.23 and 531.65 eV with FWHM ofabout 1.6 eV and 1.9 eV were observed for c(Gly-His) adsorbedon Au(111) and Cu(110) crystals, respectively (see Table 1). TheO 1s spectra of c(Phe-Pro) show peaks at 531.05 (Au(111)) and531.40 eV (Cu(110)) with FWHM of 1.6 eV and 1.35 eV, respec-tively. In summary, the O 1s binding energies are 0.3–0.4 eVlower on Au than on Cu, consistent with stronger screening ofthe molecules with the Au surface, that is, the oxygen is closerto the metal surface. This will be discussed further in the nextsection on NEXAFS.

From analysis of the Au 4f, C 1s, N 1s, and O 1s spectra, weconclude that the most significant changes between adsorptionon Cu and Au occur for the N 1s spectra. The cyclic dipeptidesbond to the Cu(110) surface via a N–Cu bond, while on theAu(111) such strong interaction was not observed.

NEXAFS spectra

While the XPS data provide information on the compositionand bonding of cyclic dipeptides on Cu and Au, the geometricalorientation of these molecules can be derived from NEXAFSdata. The technique is based on the fact that the matrix elementfor excitation of N 1s and O 1s electrons into the unoccupiedmolecular orbitals depends on the relative orientation of theelectric field vector with respect to these orbitals.33 Typical Nand O K-edge absorption spectra of c(Gly-His) and c(Phe-Pro)molecules adsorbed on Au(111) and Cu(110) surfaces are shownin Fig. 4 and 5. The peak positions and assignments are presentedin Table 2, and the present assignments are based on previouslyreported data for adsorbed biomolecules on surfaces.12,30–32

According to the building block scheme,34 the NEXAFSspectrum of a complex molecule can be understood as a super-position of its constituents. Based on the published spectra ofthe building blocks of cyclic dipeptides, namely the amino,pyrrolidine and imidazole groups, the four features A–D observedin the N K-edge NEXAFS spectra of c(Gly-His)/Au(111) (Fig. 4a,upper panel) can be assigned as shown in Table 2. The peak Aat 400.6 eV is attributed to the N 1s - p* transition forthe imino nitrogen in the IM ring, while the second peak B at401.8 eV is due to N 1s - p* transitions of the 1s electrons ofamino nitrogen in the IM and DKP rings. We also assign acontribution to peak B from s*NH, transitions, which areresolved in the gas phase35 but unresolved in the presentspectra. The energies are in agreement with published dataof histidine and histidine-containing peptides on Au(111),although in the work12 we did not include the assignment ofthe s* contribution. The two broad features C and D centred

PCCP Paper

Publ

ishe

d on

10

Febr

uary

201

4. D

ownl

oade

d by

SIN

CR

OT

RO

NE

TR

IEST

E S

C. P

.A. o

n 03

/03/

2014

13:

34:2

7.

View Article Online

Phys. Chem. Chem. Phys. This journal is© the Owner Societies 2014

at 406.8 and 413.4 eV are attributed to transitions of 1selectrons of all nitrogen atoms to s* (N–C) resonances. Theseassignments are derived from reported data on adsorbed

histidine and other amino acids and imidazole in the solidphase.12,30–32 In the case of c(Phe-Pro) only three resonancesA–C were observed (see Fig. 4a, lower panel). The lowest energy

Fig. 4 N K-edge (a) and O K-edge (b) NEXAFS spectra of c(Gly-His) and c(Phe-Pro) molecules adsorbed on Au(111), measured at GI and NI.

Fig. 5 N K-edge (a) and O K-edge (b) NEXAFS spectra of c(Gly-His) and c(Phe-Pro) adsorbed on Cu(110), measured at NI with EJ[001] (perpendicular tothe close packed copper rows) or EJ[1%10] (parallel to the close-packed rows), and GI where EJ[110] is nearly normal to the surface.

Paper PCCP

Publ

ishe

d on

10

Febr

uary

201

4. D

ownl

oade

d by

SIN

CR

OT

RO

NE

TR

IEST

E S

C. P

.A. o

n 03

/03/

2014

13:

34:2

7.

View Article Online

This journal is© the Owner Societies 2014 Phys. Chem. Chem. Phys.

peak A is associated with 1s to p* and s* transitions of nitrogenatoms in the DKP ring, while the peaks B and C are due tothe s* resonances. As the A resonance contains a mixture ofp* and s* transitions we cannot extract quantitative angulardependence.35

The O K-edge spectra of the cyclic dipeptides adsorbed onthe Au(111) surface are shown in Fig. 4b. The peaks at 532.55 eVfor c(Gly-His) and 532.35 eV for c(Pro-Phe) are attributed to theO 1s - p*CQO transition while the broad features in thehigher photon energy range are assigned to the s* resonance.These energies are in good agreement with previous NEXAFSand electron energy loss studies of amino acids and peptides inthe solid state.12,30,31,36

In contrast to the N K-edge, the O 1s - p* resonances arepure and so accurate orientation information can be extracted.The angular dependence of the p* resonance intensity varies asI p cos2 y,37 where y is the angle between the E-vector anddirection of the orbital. Using this equation, we have found thatthe tilt angle between the c(Gly-His) and c(Phe-Pro) moleculeswith respect to the Au(111) surface is B121 and B161, respec-tively, that is, almost flat.

Depending on the symmetry of the system, different levels ofinformation about the angular orientation of a molecularadsorbate can be obtained from NEXAFS data.33 The Cu(110)surface belongs to the irreducible representation C2v, so theNEXAFS spectra were measured for three geometries where theE-vector was along a principal axis: perpendicular or parallelto the close-packed copper rows, i.e. along the [001] or [1%10]direction, respectively, at normal incidence; and nearly normalto the (110) surface at grazing incidence of the light. TheN K-edge spectra of the cyclic dipeptides adsorbed on Cu(110)are shown in Fig. 5a and the spectral features also divide intop* and s* regions. The more intense sharp peaks A and B atB400.6 and B402.0 eV for both dipeptides are assigned totransitions of 1s electrons to p* resonances (see Fig. 5a).Following the assignments given for the gas phase and solidstate spectra of imidazole and histidine,11,32 these two featuresin the N K-edge NEXAFS spectra of c(Gly-His) are attributed totransitions of the two nitrogen atoms, N(imino) 1s - p* and

N(amino) 1s - p* of the IM ring. However, c(Phe-Pro) does notcontain an imino nitrogen atom. We assign the feature A in theNEXAFS spectra of c(Phe-Pro) to a chemisorption state ofnitrogen on the copper surface, related to the new feature atB398.45 eV in the N 1s photoelectron spectrum (see above).Transitions from N 1s of amino nitrogen atoms in the IM andDKP rings to the p* molecular orbital contribute to the maxi-mum B in the spectra. The broad features C and D are due tothe excitation to s* (N–C) resonances of both nitrogen atoms.The strong contribution of the N 1s electron of the pyrrolidinering of c(Phe-Pro) to the s* molecular orbital can be observedfor peak C. Such a transition was also very distinct in thespectrum of proline in the gas phase.35 The spectrum ofc(Phe-Pro) exhibits the strongest p* (A and B) resonances forE aligned in the [001] direction, weaker resonances for EJ[1%10],and weakest intensity for EJ[110] geometry. These results showthat the molecules are adsorbed at an angle larger than themagic angle (54.71) on the Cu(110) surface.

The difference in orientation and bonding also explains thedifference in thickness reported in Section 3.1. The averageeffective thicknesses were about 40% larger on Cu than on Au,and this is consistent with fairly flat-lying side groups on Au,and strongly tilted ones on Cu.

The XPS data above showed that both molecules are bondedthrough nitrogen to the surface. For steric reasons, only onenitrogen atom can bond if the molecular plane is strongly tiltedwith respect to the surface. For c(Phe-Pro), the bond must occurvia the N8 atom, as the N2 atom is too far from the surface in thisgeometry (see Fig. 1). For c(Gly-His) either the N3 or N6 atom maybond to the surface, as they are not sterically hindered, but it isnot possible for both atoms to bond. The p* resonances are moreintense for EJ[001] than for EJ[1%10], indicating that the moleculesare azimuthally oriented with their molecular plane preferentiallyparallel to the close-packed copper rows. Since the molecules aretilted, those which are bonded via nitrogen can be bondedthrough one atom only for steric reasons, as stated above.

The spectra of c(Gly-His) taken at EJ[001] and EJ[1%10] show asimilar spectral shape with the strongest p* (A and B) resonances inthe EJ[001] and EJ[1%10] geometries, and with weakest p* intensity

Table 2 Peak positions for the p* and s* resonances in the N and O NEXAFS spectra of cyclic dipeptides

Au(111) N K-edge O K-edge Cu(110) N K-edge O K-edge

c(Gly-His) NI 400.6 (p*N(imino))-A401.8 (p*N(amino), s*NH)-B406.8 (s*NC)-C413.4 (s*NC)-D

532.35 (p*CQO) EJ[001] and EJ[1%10] 400.6 (p*N(imino))-A402.0 (p*N(amino))-B406.4 (s*NC)-C413.0 (s*NC)-D

532.55 (p*CQO)

GI 400.4 (p*)401.6 (p*)406.8 (s*)413.2 (s*)

532.35 (p*) EJ[110] 400.6 (p*)402.0 (p*)406.4 (s*)413.0 (s*)

532.55 (p*)

c(Phe-Pro) NI 401.6 (s*NH, p*N(amino))-A405.6 (s*NC)-B413.2 (s*NC)-C

532.35 (p*CQO) EJ[001] and EJ[1%10] 400.4 (p*N–Cu)-A401.8 (p*, s*N(amino))-B405.0 (s*NC)-C412.8 (s*NC)-D

532.35 (p*CQO)

GI 401.6 (p*)405.4 (s*)413.2 (s*)

532.35 (p*) EJ[110] 400.2 (p*)401.8 (p*)405.0 (s*)412.8 (s*)

532.35 (p*)

PCCP Paper

Publ

ishe

d on

10

Febr

uary

201

4. D

ownl

oade

d by

SIN

CR

OT

RO

NE

TR

IEST

E S

C. P

.A. o

n 03

/03/

2014

13:

34:2

7.

View Article Online

Phys. Chem. Chem. Phys. This journal is© the Owner Societies 2014

for EJ[110], which supports an upright orientation (tilt greaterthan 54.71). However, the interpretation of this behaviour ismore complicated as the relative intensities of the p* reso-nances change for EJ[110] with respect to the other two geo-metries. This is because c(Gly-His) consists of two rings, andthe nitrogen atoms of each ring contribute to the resonances.We conclude that the rings are not parallel to one another.

In the p* region of the O K-edge spectra, resonances due tothe peptide O 1s - p* transitions were observed at 532.55 eVand 532.35 eV for c(Gly-His) and c(Phe-Pro), respectively (seeTable 2). The identification of the s* transitions is consistentwith that of the O 1s NEXAFS spectra of the dipeptidesdeposited on Au(111). The intensity ratio of the p*/s* reso-nances in the spectra supports our interpretation discussed forthe N K-edge spectra.

Conclusions

In the present work c(Gly-His) and c(Phe-Pro) assembled on goldand copper surfaces have been studied. The electronic structureand orientation of these molecules were analyzed using X-rayphotoelectron spectroscopy with synchrotron radiation and near-edge X-ray absorption spectroscopy. XPS results show chemicalbinding of the c(Gly-His) and c(Phe-Pro) adsorbates to Au(111)and Cu(110) substrates through the nitrogen atoms. In the caseof the Cu substrate, the binding was much stronger, occurringprimarily via one of the DKP nitrogen atoms and the IM ring forc(GlyHis), and dehydrogenation may have occurred.

Compared with linear Gly-His absorbed from the liquidphase on Au(111)11 there were no significant differences inthe formation of chemisorbed monolayers between c(Gly-His)dipeptides deposited on the same surface in a vacuum.

The N and O NEXAFS spectra showed a strong angulardependence allowing us to draw conclusions about the molecularorientation. For gold, the decrease of the p* resonance intensityat NI indicates that the molecular plane of the c(Gly-His) andc(Phe-Pro) peptides are closer and parallel to the Au(111) surface.This may imply some weak p bonding with the conjugatedelectron system of the DKP ring. However the same moleculesare predominantly in an upright geometry on Cu(110). Thisinteraction occurs due to chemical bonding through one of thenitrogen atoms of the DKP ring.

The present study indicates that even with the increasingcomplexity of peptides, it is possible to gain useful informationabout chemical bonding between an adsorbed molecule and asurface, and the orientation of functional groups with respect tothe surface. This possibility is encouraging for further investi-gations of more complex biomolecules, using both surfacesensitive techniques.

Acknowledgements

We acknowledge funding from the European Community’sSeventh Framework Programme (FP7/2007-2013) under grantagreement No. 226716. We gratefully acknowledge the assistance

of our colleagues at Elettra for providing good quality synchro-tron light. The Materials Science Beamline is supported by theMinistry of Education of the Czech Republic under Grant No.LG12003. S.P. acknowledges the Division of Chemical Sciences,Geosciences and Biosciences, Basic Energy Sciences, Office ofScience, United States Department of Energy (DOE) throughGrant No. DE-FC02-04ER15533 (NDRL no: 4873).

Notes and references

1 Z.-C. Xing, Y. Chang and I.-K. Kang, Sci. Technol. Adv. Mater.,2010, 11, 014101.

2 J. D. Andrade, V. Hlady and A. P. Wei, Pure Appl. Chem.,1992, 64, 1777.

3 J. Israelachvili, Intermolecular and surface forces, AcademicPress, New York, 1992, p. 450.

4 J. V. Barth, G. Costantini and K. Kern, Nature, 2005,437, 671.

5 A. Kuhnle, T. R. Linderoth, B. Hammer and F. Besenbacher,Nature, 2002, 415, 891.

6 R. Otero, M. Schock, L. M. Molina, E. Lægsgaard, I. Stensgaard,B. Hammer and F. Besenbacher, Angew. Chem., 2005, 117, 231,(Angew. Chem., Int. Ed., 2005, 44, 2270).

7 Y. Zubavichus, A. Shaporenko, M. Grunze and M. Zharnikov,Nucl. Instrum. Methods Phys. Res., Sect. A, 2009, 603, 111;Y. Zubavichus, A. Shaporenko, M. Grunze and M. Zharnikov,J. Phys. Chem. B, 2008, 112, 4478; Y. Zubavichus, M. Zharnikov,A. Schaporenko and M. Grunze, J. Electron Spectrosc. Relat.Phenom., 2004, 134, 25.

8 A. Valle, V. Humblot and C.-M. Pradier, Acc. Chem. Res.,2010, 43, 1297.

9 A. Valle, V. Humblot, C. Methivier and C.-M. Pradier, J. Phys.Chem. C, 2009, 113, 9336.

10 C. Methivier, V. Lebec, J. Landoulsi and C.-M. Pradier,J. Phys. Chem. C, 2011, 115, 4041.

11 V. Feyer, O. Plekan, T. Skala, V. Chab, V. Matolın andK. C. Prince, J. Phys. Chem. B, 2008, 112, 13655.

12 V. Feyer, O. Plekan, N. Tsud, V. Chab, V. Matolın andK. C. Prince, Langmuir, 2010, 26, 8606.

13 V. Feyer, O. Plekan, N. Tsud, V. Lyamayev, V. Chab,V. Matolın, K. C. Prince and V. Carravetta, J. Phys.Chem. C, 2010, 114, 10922.

14 V. Feyer, O. Plekan, S. Ptasinska, M. Iakhnenko, N. Tsud andK. C. Prince, J. Phys. Chem., 2012, 116, 22960.

15 C. Prasad, Peptides, 1995, 16, 151.16 R. S. McDowell and T. R. Gadek, J. Am. Chem. Soc., 1992,

114, 9245.17 D. D. Smith, J. Slaninova and V. J. Hruby, J. Med. Chem.,

1992, 35, 1558.18 P. J. Milne, A. L. Hunt, K. Rostoll, J. J. van der Walt and

C. J. Graz, J. Pharm. Pharmacol., 1998, 50, 1331.19 A. P. W. Arachchilage, F. Wang, V. Feyer, O. Plekan and

K. C. Prince, J. Chem. Phys., 2010, 133, 174319.20 A. P. W. Arachchilage, F. Wang, V. Feyer, O. Plekan and K. C.

Prince, J. Chem. Phys., 2012, 136, 124301.

Paper PCCP

Publ

ishe

d on

10

Febr

uary

201

4. D

ownl

oade

d by

SIN

CR

OT

RO

NE

TR

IEST

E S

C. P

.A. o

n 03

/03/

2014

13:

34:2

7.

View Article Online

This journal is© the Owner Societies 2014 Phys. Chem. Chem. Phys.

21 R. Degeilh and R. E. Marsh, Acta Crystallogr., 1959,12, 1007.

22 R. Vasina, V. Kolarık, P. Dolezel, M. Mynar, M. Vondracek,V. Chab, J. Slezak, C. Comicioli and K. C. Prince, Nucl.Instrum. Methods Phys. Res., Sect. A, 2001, 467–468, 561.

23 http://www.bachem.com/.24 M. P. Seah and W. A. Dench, Surf. Interface Anal., 1979, 1, 2.25 http://www.chemspider.com.26 K. Heister, M. Zharnikov, M. Grunze and L. S. O. Johansson,

J. Phys. Chem. B, 2001, 105, 4058.27 F. Reinert and G. Nicolay, Appl. Phys. A: Mater. Sci. Process.,

2001, 78, 817.28 A. Nuber, M. Higashiguchi, F. Forster, P. Blaha, K. Shimada

and F. Reinert, Phys. Rev. B: Condens. Matter Mater. Phys.,2008, 78, 195412.

29 Y. Zubavichus, M. Zharnikov, Y. Yang, O. Fuchs, C. Heske,E. Umbach, G. Tzvetkov, F. P. Netzer and M. Grunze, J. Phys.Chem. B, 2005, 109, 884.

30 J. Jones, L. B. Jones, F. Thibault-Starzyk, E. A. Seddon,R. Raval, S. J. Jenkins and G. Held, Surf. Sci., 2006, 600, 1924.

31 J. Hasselstrom, O. Karis, M. Weinelt, N. Wassdahl,A. Nilsson, M. Nyberg, L. G. M. Pettersson, M. G. Samantand J. Stohr, Surf. Sci., 1998, 407, 221.

32 E. Apen, A. P. Hitchcock and J. L. Gland, J. Phys. Chem.,1993, 97, 6859.

33 M. Neuber, M. Zharnikov, J. Walz and M. Grunze, Surf. Rev.Lett., 1999, 6, 53.

34 L. G. M. Pettersson, H. Ågren, B. L. Schurmann, A. Lippitzand W. E. S. Unger, Int. J. Quantum Chem., 1997, 63, 749.

35 O. Plekan, V. Feyer, R. Richter, M. Coreno, M. de Simone,K. C. Prince and V. Carravetta, J. Electron Spectrosc. Relat.Phenom., 2007, 155, 47.

36 M. L. Gordon, G. Cooper, C. Morin, T. Araki, C. C. Turci,K. Kaznatcheev and A. P. Hitchcock, J. Phys. Chem. A, 2003,107, 6144.

37 J. Stohr, NEXAFS Spectroscopy, Springer-Verlag, Berlin, 1992.

PCCP Paper

Publ

ishe

d on

10

Febr

uary

201

4. D

ownl

oade

d by

SIN

CR

OT

RO

NE

TR

IEST

E S

C. P

.A. o

n 03

/03/

2014

13:

34:2

7.

View Article Online